The present invention relates to a new phosphatase enzyme. In particular, the present invention relates to the discovery of a novel heat-labile phosphatase enzyme from the filamentous fungus Aspergillus niger. This new phosphatase enzyme is used as a highly-specific reagent for the hydrolytic removal of terminal phosphate groups from linear DNA molecules during one-step radioactive end-labeling procedures, as well as during preparation of linear DNA molecules for use in molecular cloning assays.
In the following discussion, a number of citations from professional journals are included for the convenience of the reader. While these citations will more fully describe the state of the art to which the present invention pertains, the inclusion of these citations is not intended to be an admission that any of the cited publications represent prior art with respect to the present invention.
To place this new microbe-derived phosphatase enzyme in perspective, it will be helpful to provide some background for the molecular processes in which phosphate groups are transferred among cytoplasmic constituents in a normal cell. In the action of many polypeptide hormones, a critically-important cellular mediator is the "second messenger" protein called cyclic adenosine monophosphate (cAMP), which is formed from adenosine triphosphate (ATP) by the enzyme adenylate cyclase; this latter enzyme is bound to the cytoplasmic side of plasma membranes (see Watson et al., Molecular Biology of the Gene (4th Edition), The Benjamin/Cummings Publishing Company, Menlo Park, Calif., 1987). After its synthesis, cAMP works by stimulating the activity of cAMP-dependent enzymes called protein kinases. Protein kinases are enzymes that transfer the high-energy terminal phosphate group of ATP to specific amino acids (serine, threonine, or tyrosine residues) on target proteins. Phosphorylation (i.e., the process of adding a phosphate group to a protein) alters the enzymatic activities of these target proteins, and, depending on the particular enzymes involved and the location of the added phosphate moiety, can either raise or lower their functional activities.
In a like manner, removal of a phosphate group from a protein (a process known as "dephosphorylation") can also greatly modify the functions and activities of certain biological molecules. Dephosphorylation occurs by a process of "hydrolysis" in which the phosphate group is catalytically broken away ("lysed") from a parent molecule by the enzymatic addition of a water molecule to the parent molecule. This ongoing and cyclic process of phosphorylation followed by dephosphorylation, as well as dephosphorylation followed by rephosphorylation, are essential processes in the energy-efficient functioning of all living cells.
The dephosphorylation of a linear DNA molecule by the removal of the highly-reactive terminal 5'-phosphate group is an essential step in a number of molecular cloning protocols. Removal of the highly-reactive terminal phosphate prevents the linear DNA molecule from spontaneously ligating to the 3'-hydroxyl group at the opposite end of the same molecule, or to terminal hydroxyl groups on other reactive "bystander" DNA molecules in the same reaction mixture. In addition to this important "house-keeping" function, the dephosphorylation reaction is used by research investigators to prepare the reactive 5'-ends of a linear DNA molecule for subsequent radioactive end-labeling in the presence of polynucleotide kinase and [gamma-.sup.32 P]-ATp, as will be discussed more fully hereinafter.
Currently, the enzyme most widely used in molecular biology protocols for the removal of the terminal 5'-end phosphates from DNA molecules is calf intestine alkaline phosphatase (as discussed in the recent volume by Maniatis T., Fritsch E. F., and Sambrook J. (editors): Molecular Cloning: A Laboratory Manual; New York: Cold Spring Harbor Laboratory, pages 133-134, 1982). When used in any of a variety of molecular cloning procedures, this bovine-derived enzyme has the advantage over other previously-used phosphatases in that it can be completely denatured (with total loss of activity) by heating the reaction mixture to 68.degree. C. in the presence of an additional denaturing agent such as the negatively-charged detergent sodium dodecyl sulfate (SDS). Under these conditions, the calf intestine alkaline phosphatase is completely destroyed without denaturing the DNA in the reaction mixture. This is important because the native calf enzyme has the capacity to react with a wide variety of phosphate-bearing substrates, including the energy-rich molecule adenosine triphosphate (ATP). Because of this capacity to react with ATP, it is absolutely necessary to inactivate or remove the calf phosphatase after the dephosphorylation reaction is complete, in order to prevent its interfering with phosphate transfer from ATP in the subsequent reaction steps of an end-labeling protocol.
The fairly low inactivation temperature of 68.degree. C. is an important factor in the current selection of calf intestine alkaline phosphatase for use in molecular cloning and end-labeling protocols. Other alkaline phosphatases, derived from such sources as the bacterium Escherichia coli, can only be inactivated by boiling the reaction mixture in which that enzyme is contained. Such harsh temperatures (at least 100.degree. C.) are likely to denature more than just the phosphatase enzyme, and may cause irreversible damage to the DNA as well. Furthermore, it is not clear that boiling is sufficient to completely inactivate the E. coli phosphatase, making the use of this microbial enzyme even less attractive.
While working with the calf intestine alkaline phosphatase does present some significant advantages over using other phosphatase enzymes, there are disadvantages. For example, the need for the combination of heating the reaction mixture to 68.degree. C. and using SDS to inactivate the calf enzyme is cumbersome. In radioactive end-labeling procedures, for example, use of these procedures necessitates a multiplicity of reaction steps in order to eliminate the enzyme. The DNA must be precipitated from the reaction mixture, washed, and then re-isolated free of the enzyme before the DNA is used as target substrate for reactivity with other phosphate-bearing molecules in subsequent reaction steps. These multiple steps of heating, precipitating, washing and re-isolating DNA molecules are a real disadvantage in studies in which such factors as time, or temperature (or both) are critical.
Additional disadvantages which are evident when working with the calf-derived enzyme, as well as with the E. coli-derived phosphatase, are their broad reaction specificities (i.e., both have the capacity to dephosphorylate more than just DNA molecules). Furthermore, it is clear that enzymes derived from mammalian sources are not as convenient to obtain as they are from microbial sources; accordingly, mammalian-derived enzymes have associated with them certain economic disadvantages, which, depending on the enzyme source, can be very significant. Consequently, there is a need for finding an alternate source (preferably microbial) for phosphatase enzymes. This has been the objective of much research.
An important microbial source which has been well studied over the years is the filamentous fungus Aspergillus niger (hereinafter "A. niger"). Several phosphatase enzymes have been isolated from this microbial organism, and have been found to exhibit a broad substrate specificity (not unlike that of calf intestine alkaline phosphatase). With regard to functional characteristics, they have been generally categorized as being either "acid" or "alkaline" phosphatases, i.e., categorized according to the pH value at which their enzymatic function is optimal in the hydrolysis reaction associated with the dephosphorylation process: pH 2.5 to 5 and pH 8.5 to 9.5, respectively. Two reports have suggested the presence of as many as five acid phosphatase activities (i.e., pH 2.5 to 5.0) in extracts of A. niger (Komano T, Plant Cell Physiol. 16: 643-658, 1975; Pathak and Sreenivasan, Arch. Biochem. Biophys. 59: 366-372, 1955); two other reports have indicated the presence of several alkaline phosphatase activities from A. niger (functional pH optima from pH 8.5 to 10) (Rokosu and Uadia, Int. J. Biochem. 11: 541-544, 1980; Ramaswamy and Bheemeswar, Experientia 32: 852-853, 1976). These alkaline phosphatases, like the calf intestine alkaline phosphatase, have also been reported to exhibit a broad hydrolytic reactivity on substrates which include sugar phosphates, nucleotides such as adenosine-5'-phosphate, small synthetic substrates such as 4-nitrophenylphosphate, and inorganic pyrophosphate (P.about.P).
Phosphatase enzymes with a much more limited substrate specificity have also been found in microorganisms. Examples of such restrictive enzyme activity are the phosphomonoester hydrolases. One such enzyme is 4-nitrophenylphosphatase, which is highly specific for synthetic 4-nitrophenylphosphate (4-NPP) as a substrate. While this small substrate molecule is chromogenic before it is hydrolysed by the phosphatase enzyme, its chromophore is revealed only after hydrolysis, and this is shown by a significant increase in the absorption of blue light by the hydrolysed substrate.
Several distinct forms of this 4-nitrophenylphosphatase enzyme have been isolated from cells of the yeast Saccharomyces cerevisiae (Attias and Bonnet, Biochim. Biophys. Acta 268: 422-430, 1972), and have been found to function best in an environment having a pH optima between pH 8.0 and 8.5. Further characterization of these yeast-derived enzymes has shown that their functional activities are modified by the presence of certain divalent cations; such information is very important when preparing culture media in which to grow the microorganisms and to perform the test reactions. For example, these yeast-derived phosphatases are strongly activated by magnesium (Mg.sup.2+) ions, and are inhibited by zinc (Zn.sup.2+) ions.
The novel phosphatase enzyme of the present invention was discovered in cultures of A. niger. A 4-nitrophenylphosphatase activity was unexpectedly found in extracts of homogenized filamentous structures known as "mycelia." The 4-nitrophenylphosphatase extracted from these A. niger mycelia exhibited optimal enzymatic activity in a neutral-to-slightly alkaline pH environment (i.e., about pH 7.0 to about pH 8.5). Similar to the S. cerevisiae enzyme noted above, the A. niger phosphatase activity is markedly stimulated by magnesium ions, and is inhibited by cations of zinc. While being highly specific for 4-NPP as a synthetic substrate, the neutral A. niger phosphatase has physical and functional characteristics which distinguish it from the other phosphatase enzymes from calf, E. coli, and A. niger. For example, the new A. niger enzyme exhibits a unique and remarkable substrate specificity; it will interact with the 5'-terminal phosphate of a linear and polymeric DNA molecule, but it unexpectedly will not interact with the terminal phosphate group of a monomeric ATP molecule which may simultaneously be present as a phosphate donor in the same reaction mixture. This subtrate specificity is both remarkable and unexpected.
In stark contrast to the other phosphatases mentioned above, this new A. niger phosphatase is very heat labile, being completely inactivated by heating to about 50.degree. C. No denaturing agents, such as the detergent SDS, are needed to facilitate inactivation of this enzyme, as is necessary with the calf-derived alkaline phosphatase. It is very simple, therefore, to destroy the functional activity of this A. niger phosphatase (as is especially necessary when the dephosphorylated DNA substrate is to be subsequently used in a molecular cloning procedure) without risking damage or change to any of the other molecules contained in the reaction mixture.
Furthermore, this A. niger phosphatase functions best in a reaction environment which is neutral to slightly alkaline (i.e., between about pH 7.0 to 8.5). This is in significant contrast to the other microbial phosphatases tested which require either strongly acid (pH 2.5 to 5.5) or strongly basic conditions (pH 8.5 to 10) for optimal functional activity.
The novelty of this new A. niger phosphatase enzyme will become apparent in the following discussion.